XIAP is a central apoptosis regulator and inhibits apoptosis by binding to and inhibition of effectors caspase-3/-7 and an initiator caspase-9 through its BIR2 and BIR3 domains, respectively. Smac protein in its dimeric form effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. Based on the proposed model for the mechanism of Smac protein we had designed and synthesized cyclic peptide containing two-site interaction N-terminal Smac mimetic tetra peptide binding motif, named as PSmac21.
 Using a series of experiments, fluorescence polarization based binding assay, caspase activity assay, gel filtration chromatography and X-ray study we have characterized PSmac21 on biochemical, structural and functional levels. Our results showed that PSmac21 interacts with XIAP constructs with very high affinity and forms complexes with different stoichiometric ratio. PSmac21 binds to XIAP containing both BIR2 and BIR3 domains with an IC50 of 4 nM, being > 130 and > 2,500 times more potent than its linear counterpart tetrapeptide and the natural Smac AVPI peptide, respectively. Gel filtration experiments with Bir3 domain showed that PSmac21 forms a 1:2 stoichiometric complex, while in the presence of XIAP protein containing both Bir2 and Bir3 domains forms a 1:1 stoichiometric complex. These gel filtration results indicate that the dimerization of the Bir3 is propagated via the bivalent nature of the cyclic peptide, PSmac21, while with the XIAP protein containing both domains, PSmac21can simultaneously interact with the Bir2 and Bir3 domains from a single protein. Interestingly, in the presence of XIAP protein containing both Bir2 and Bir3 domains, but binding to the Bir2 domain was knocked out by mutation, Bir2(E219R)-Bir3, PSmac21causes dimerization of this protein, similar as in the presence of Bir3 only, indicating that Bir2 domain is involved in the interaction with PSmac21. This interaction not only possesses high binding affinity because of avidity, but most important PSmac21 can efficiently antagonize inhibition of effector and initiator caspases by XIAP containing both Bir2 and Bir3 domains in both, cell-free functional and cell-based assays. Importantly, PSmac21 inhibits cell growth in MDA-MB-231 breast cancer cells, indicating that this cyclic peptide is cell-permeable.
 We have determined the crystal structure of PSmac21 in a complex with XIAP Bir3 to 2.1 Å resolution. This high resolution crystal structure clearly shows that cyclic peptide PSmac21 induces homodimerization of XIAP Bir3 and provides a structural basis for the cooperative binding of one PSmac21 to two XIAP Bir3 molecules. Based on our crystal structure we have constructed a structural model between Bir2-Bir3 and PSmac21. This model provides structural information for designing of novel bivalent Smac mimetics inhibitors targeting both domains in XIAP. Our model further suggests small molecule bivalent Smac mimetics may be used as potent inhibitors mimicking the function of the Smac protein against the full length XIAP protein.

AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics-- Oct 22-26, 2007; San Francisco, CA